The present invention relates to compositions and methods for inhibiting activity of multimeric enzymes. In particular, it relates to inhibition of proteases by formation of dysfunctional protease multimers.
Protein structure is typically discussed in terms of four levels. The primary structure is the amino acid sequence; the secondary structure is any regular local structure of linear segment, such as an .alpha.-helix; the tertiary structure is the overall topology of the folded polypeptide chain; and the quaternary structure is the aggregation of single polypeptides or subunits to form a functional molecule.
Many proteins exist as assemblies of two or more polypeptide chains, which may be identical or different. Complex interactions between the subunits are required to produce a functional protein. For example, multimeric enzymes can be rendered inactive, if the interaction of the monomers is disrupted.
Multimeric enzymes of particular interest to the present invention are multimeric proteases. Inhibition of protease activity may be useful in a number of contexts. For example, inhibition of retroviral proteases, which are critical to retroviral maturation and infectivity, can be used to inhibit retroviral infection.
Retroviruses are those viruses which have a single stranded RNA genome, a lipid envelope, and encode an RNA-dependent deoxyribonucleic acid (DNA) polymerase, known as reverse transcriptase. During their life cycle, the RNA genome is reverse transcribed into a DNA copy which is integrated into the genome of the host cell. A number of retroviruses cause disease states in humans. These include the lentiviruses, human immunodeficiency viruses (HIV-1 and HIV-2), which cause acquired immune deficiency syndrome (AIDS), and the oncoviruses, human T-cell lymphotrophic viruses I and II which cause T cell leukemias.
Retroviruses, such as HIV-1, encode aspartic proteases that process polyprotein precursors into viral structural proteins and replicative enzymes that are essential for viral proliferation. Autoprocessing of the protease from the gag/pol polyprotein precursor results in the release of the protease and the generation of mature structural and enzymatic proteins derived from the gag and gag/pol polyproteins.
Studies of the crystal structure of HIV proteases (Navia, et al, Nature 337:615-620 (1989); Wlodawer, et al., Science 245:616-621 (1989)) have confirmed the homodimeric nature of these enzymes. Assembly of the two HIV protease monomers results in a dimer of approximately 22KD and generates an active site at the interface of the subunits.
These X-ray crystallographic studies have also defined regions of interaction between the monomers. Each monomer contributes half of the active site, which includes two catalytic aspartic acids as well as threonine/serine and glycine residues which are conserved among all aspartyl proteases for their structural role in maintaining active site geometry. The two N-termini and two C-termini of the individual monomers form .beta.-strands that interdigitate to create a four-stranded anti-parallel .beta.-sheet. These interactions appear to be a major stabilizing force in the enzyme and contribute over 50% of the inter-subunit contacts and hydrogen bonds. Dimer formation generates not only the catalytic center, but also the extended substrate binding pocket.
Since viral proteolytic activity is essential for the generation of infectious virus particles in HIV and related retroviruses, therapeutic intervention for HIV-1 and HIV-2 has targeted the HIV protease. Small molecules have been developed as inhibitors and are currently undergoing clinical trials as antiviral agents. In clinical trials however, resistance to these inhibitors is being observed. Thus, new approaches for inhibiting retroviral proteases, in particular HIV-1 protease, are an important therapeutic goal in the treatment of retroviral infections. The present invention addresses these and other needs.